While methotrexate has been in clinical use for a number of years as an antitumor agent, the recent introduction of high dose schedules followed by citrovorum factor rescue in the treatment of solid tumors has resulted in renewed interest in this agent, and has revealed that our knowledge of the clinical pharmacology of this and related folate antagonists is still incomplete. During the past year, unequivocal evidence has been obtained by field desorption mass spectrometry and by proton NMR spectrometry of the structure of the major metabolite of methotrexate, 7-hydroxymethotrexate.